Short interfering RNAs (siRNAs) (also known as short interfering nucleic acid (siNA) molecules) that mediate sequence-specific gene silencing through RNA interference (RNAi) are widely used research tools that have potentially broad therapeutic applications (Dorsett et al, Nature Reviews, 3:318-329 (2004); Soutschek et al., Nature, 432:173-178 (2004)). siRNAs mediate gene silencing through their incorporation into RISC, a component of the RNAi machinery, that initiates the site specific cleavage of homologous mRNA (Dorsett et al., supra). siRNAs can be generated from expression vectors producing short hairpin RNAs that are subsequently processed to form duplex siRNA. Alternatively, siRNAs can be manufactured chemically. Synthetic siRNAs offer a number of advantages, since they permit chemical modifications designed to improve critical parameters such as nuclease stability and pharmacokinetic behavior.
The mammalian innate immune system has evolved mechanisms for recognizing a number of nucleic acid species that are signatures of potential pathogens. Toll-like Receptors (TLRs) (Takeda et al, Ann. Rev. Immunol., 21:335-76 (2003)) have been identified that recognize dsRNA (TLR3) (Alexopoulou et al., Nature, 413:732-738 (2001)), ssRNA (TLR7 and TLR8) (Heil et al., Science, 303:1526-29 (2004); Diebold et al., Science, 303:1529-31 (2004); Lund et al., Proc. Natl. Acad. Sci. U.S.A., 101:5598-5603 (2004)), and CpG DNA (TLR9) (Krieg, Annu. Rev. Immunol., 20:709-760 (2002)) in humans and mice. Common features of these nucleic acid sensing TLRs is their intracellular localization and the induction of Type I interferons such as IFN-α upon stimulation. Activation of the innate immune system causes the rapid production of pro-inflammatory cytokines together with Type I and Type II interferons that orchestrate the developing immune response. This reaction can be associated with acute symptoms of toxicity and inflammation that in severe cases can develop into systemic toxic-shock like syndromes. Unless anticipated and well understood, these phenomena have the potential to limit the utility of siRNA-based therapeutics.
While it has been recently reported that naked, unformulated siRNA is incapable of eliciting an interferon response in mice (Heidel et al., Nat. Biotechnol., 22:1579-1582 (2004)), the pharmacokinetic behavior of such molecules is poor and may limit exposure to critical cell types in vivo. The poor pharmacokinetic properties of naked, unformulated siRNA are likely associated with nuclease degradation and rapid renal clearance. The administration of siRNA combined with delivery vehicles will likely increase the exposure of innate immune cells to siRNA and therefore enhance their potential to stimulate an immune response. In fact, synthetic siRNA, when associated with either lipidic or non-lipidic delivery systems, can activate potent interferon and inflammatory cytokine responses in human blood in vitro or when administered to mice (Judge et al, Nat. Biotechnol., 23:457-462 (2005)). This activity is dependent on the sequence of the siRNA duplex and likely involves the engagement of TLR7 (Judge et al., supra; Hornung et al., Nat. Med., 11:263-270 (2005)).
Thus, there is a strong need in the art for siRNA molecules that abrogate the immunostimulatory activity of siRNA without having a negative impact on RNAi activity. The present invention addresses this and other needs.